The invention is directed to novel osteoregulin polypeptides, which play a role in regulating bone homeostasis, adiposity, and the calcification of atherosclerotic plaques. The invention also features screening assays to identify modulators of osteoregulin activity as well as methods of treating mammals for diseases or disorders associated with osteoregulin activity.
Bone is a highly dynamic tissue that undergoes continual processes of remodeling and modeling (Parfitt, J. Cellular Biochemistry 55: 273-86, 1994). In the growing skeleton, the amount of mineralized bone formed exceeds the amount lost through resorption, whereas in the mature adult, bone loss and bone formation are equivalent, thereby preserving the integrity of the skeleton. Under certain conditions such as aging, postmenopausal estrogen deficiency, or prolonged steroid treatment, the amount of bone formed is not sufficient to compensate for the quantity lost by resorption. Over time, this imbalance results in reduced bone mass and compromises the structural integrity of the skeleton.
Bone remodeling is a very complex process of tightly coordinated action by the bone resorbing osteoclasts and the bone forming osteoblasts. Osteoblasts are derived from a mesenchymal cell lineage and are responsible for the formation of new bone matrix in their differentiated state (Wlodarski, Clinical Orthopaedics and Related Research 276: 93, 1990). In addition, factors produced by osteoblasts regulate the formation of osteoclasts and osteoctastic bone resorbing activity in response to endocrine signals such as parathyroid hormone and vitamin D. It has been postulated that the bone loss associated with aging is a result of a defect in the osteoblast cell lineage (Rodriguez et al., J. of Cellular Biochemistry 75: 414-423, 1999; Erdmann et al., Mechanisms of Aging and Development 110: 73-85, 1999; Roholl et al., J. of Bone and Mineral Research 9: 355-66, 1994; Katzburg et al., Bone 25: 667-673, 1999). Either the mesenchymal precursor population is insufficient or has lost the capacity to proliferate and differentiate into sufficient numbers of functioning osteoblasts.
Osteoblasts progress through a 3 stage process of differentiation: proliferation, matrix maturation, and mineralization (Aubin, Journal of Cellular Biochemistry 72: 396-410, 1999; Stein and Lian, Endocrine Reviews 14: 424-42, 1993; Malaval et al., J. of Cellular Biochemistry 74: 616-27, 1999). During this differentiation process, a well characterized temporal and spatial expression pattern of extracellular bone matrix proteins and other genes occurs (Malaval et al., J. of Cellular Biochemistry 74: 616-27, 1999; Owen et al., J. of Cellular Physiology 143: 420-30, 1990; Ingram et al., J. of Bone and Mineral Research 8: 1019-29, 1993). The bone matrix is composed primarily of Type I collagen which forms the extracellular structural component for the deposition of mineral forming hydroxyapatite. Osteoblasts also secrete non-collagenous proteins into the extracellular matrix (Robey, Connective Tissue Research 35: 131-6, 1996; Boskey, Connective Tissue Research 35: 357-63, 1996). The non-collagenous proteins include proteoglycans, sulfated glycoproteins, highly phosphorylated RGD-motif proteins, and proteins modified to contain gla amino acid residues. Examples include biglycan, osteonectin, bone sialoprotein, osteopontin, and osteocalcin. The exact function of many of these proteins have not yet been delineated although most evidence supports their role in promoting mineralization events (Robey, Connective Tissue Research 35: 131-6, 1996). An exception is the gla-peptide, osteocalcin, which has been shown to be a negative regulator of bone formation by gene knockout technology (Ducy et al.; Nature 382: 448-52, 1996).
The progression of osteoblast differentiation has been modeled in cell culture using primary calvarial cells or bone marrow cells. Bone marrow contains pluripotent stem cells of the adipocytic, osteoblastic, fibroblastic, and hematopoetic cell lineage (Owen et al., J. of Cellular Physiology 143: 420-30, 1990; Beresford, J. of Cell Science 102: 341-51, 1992: Herbertson and Aubin, Bone 21: 491-500, 1997). Bone marrow from rats, mice, and humans has been shown to contain osteoprogenitor cells that proliferate and can be induced to differentiate into osteoblastic cells. Rat and human cultures require a differentiation agent such as dexamethasone whereas mouse-derived cells can differentiate in the absence of dexamethasone (Rickard et al., J. of Bone and Mineral Research 11: 312-24, 1996; Maniatopoulos et al., Cell and Tissue Research 254: 317-30, 1988; Chen et al., Endocrinology 112: 1739-45, 1983). In vitro differentiated osteoblasts display the capacity to secrete noncollagenous proteins into the extracellular matrix in a temporally regulated manner which may indicate a regulatory function for each of these proteins in the mineralization process (Yao et al., J. of Bone and Mineral Research 9: 231-40, 1994). Furthermore, differentiated bone marrow cultures have the capacity to facilitate the deposition of matrix and the formation of hydroxyapatite mineral when grown in the presence of a phosphate source such as xcex2-glycerophosphate. These properties have made the differentiation of bone marrow cells a useful model to investigate the mechanisms of bone remodeling and osteoblast function.
Osteoporosis accounts for approximately 700,000 fractures per year in the United States alone, and osteoporotic fractures are linked to significant death and is morbidity in the aged population. Therefore, there is a clear need to further understand the process of bone remodeling, both in normal and pathological states, in order to develop therapeutic agents to prevent, reduce, or reverse bone loss associated with osteoporosis or other bone-related disorders.
We sequenced a novel cDNA transcript expressed specifically in rat osteoblasts and osteocytes that encodes a 45 kDa polypeptide; and we have also identified the mouse and human forms. Our characterization revealed the protein to be a secreted, RGD motif containing protein with a limited homology to dmp1, an extracellular matrix protein present in bone and teeth (Roholl et al., J. of Bone and Mineral Research 9: 355-66, 1994; Katzburg et al., Bone 25: 667-673, 1999). Thus, we have designated this mammalian protein xe2x80x9costeoregulin.xe2x80x9d Further studies of osteoregulin expression patterns and function (as further described in the detailed description) have confirmed that osteoregulin plays an important role in controlling bone homeostasis, adipose regulation, and the calcification of atherosclerotic plaques.
The invention features novel osteoregulin polypeptides, the nucleotide sequences that encode them, expression vectors containing these osteoregulin sequences, and transgenic hosts which have been genetically modified to express the osteoregulins of the invention.
Another feature of the invention is non-human mammals and animal cells that have been genetically-modified to disrupt one or both copies of an endogenous osteoregulin gene. Studies of genetically-modified mice that are homozygous or heterozygous for the osteoregulin gene disruption demonstrate that the absence or reduction of osteoregulin gene expression results in increased bone mass, increased bone mineralization, increased bone formation, and an increase in adiposity in females. These phenotypes indicate that osteoregulin functions as a negative regulator of bone mass/density and adiposity. This role in bone homeostasis is further supported by the significant expression of osteoregulin in bone tissue as well as osteoregulin""s similarities to other proteins that play a role in regulating bone function.
Given the discovered function of osteoregulins as negative regulators of bone formation, bone density, bone mineralization, as well as its role in adiposity and plaque calcification, the present invention also features screening assays to identify agents that modulate osteoregulin activity or gene expression. Such agents are useful for administering to mammals, preferably humans, for the treatment of bone disorders, such as osteoporosis, to stimulate bone repair or regeneration, and to treat disorders related to adiposity or the calcification of atherosclerotic plaques.
In its first aspect, the invention features an isolated or purified polypeptide, or a heterologous polypeptide, wherein the polypeptide has osteoregulin activity and contains: the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 34, or 46; an amino acid sequence encoded by a polynucleotide which will hybridize under highly stringent conditions with a probe having the complement of the coding sequence shown in SEQ ID NO: 1, 3, 5, 7, 33; or 45; or an amino acid sequence having at least 90% identity, more preferably, 95% identity, to an amino acid sequence containing SEQ ID NO: 2, 4, 6, 8, 34, or 46. Preferably, the polypeptide is mammalian and/or naturally occurring.
In a second, related aspect, the invention features an isolated or purified polynucleotide containing: the coding sequence of SEQ ID NO: 1, 3, 5, 7, 33, or 45; a sequence encoding any of the polypeptides above, preferably SEQ ID NO: 2, 4, 6, 8, 34, or 46; or a sequence which hybridizes, under highly stringent conditions, to a probe, preferably to the full length of a probe, having the complement of any of the above-mentioned polynucleotides. Preferably, the sequence hybridizes to the complement of the coding sequence shown in SEQ ID NO: 1, 3, 5, 7, 33, or 45, or the sequence is present in a mammalian cDNA library.
The invention also features an antibody that selectively binds to a polypeptide of the invention, preferably SEQ ID NO: 2, 4, 6, 8, 34, or 46, a vector containing a polynucleotide of the invention, preferably SEQ ID NO: 1, 3, 5, 7, 33, or 45, and a transgenic host expressing a polynucleotide of the invention, preferably the coding sequence shown in SEQ ID NO: 1, 3, 5, 7, 33, or 45. The preferred host is a transfected mammalian host cell or a transgenic mammal (e.g., mouse, rat, pig, sheep, monkey).
Another aspect of the invention is a genetically-modified, non-human mammal, wherein the modification results in a functionally disrupted osteoregulin gene. The mammal may be heterozygous for the modification, or homozygous for the modification. Preferably, the mammal is a rodent, more preferably, a mouse. In a related aspect, the invention also provides a genetically-modified animal cell, wherein the modification comprises a functionally disrupted osteoregulin gene. The cell is heterozygous or homozygous for the modification. Preferably, the animal cell is an embryonic stem (ES) cell or an ES-like cell, the cell is human, or the cell is murine.
A method of screening for an agent that modulates osteoregulin activity (e.g., affecting the regulation of bone mass, bone density, adiposity, vascular flexibility, and/or atherosclerotic plaque calcification) is an additional feature of the invention, the method comprises contacting an agent with an osteoregulin polypeptide and measuring the activity of the osteoregulin, wherein a difference between the osteoregulin activity in the presence of the agent and in the absence of the agent is indicative that the agent modulates the activity.
Also featured is a method of screening for an agent that modulates any of the above-mentioned osteoregulin activities by regulating osteoregulin expression, the method comprises contacting an agent with a cell containing a nucleotide sequence containing an osteoregulin gene regulatory element (e.g., an osteoregulin promoter sequence) operably linked to a coding sequence, and measuring the expression of the coding sequence, wherein a difference between the expression in the presence of the agent and in the absence of the agent is indicative that the agent modulates osteoregulin expression.
The invention also provides a method of treating a mammal to regulate bone mass and/or density, adiposity, vascular flexibility, and/or atherosclerotic plaque calcification, the method comprises administering an osteoregulin modulator to the mammal. Preferably, the modulator is an osteoregulin antagonist and is administered to increase bone mass and/or bone density, the modulator is an osteoregulin agonist and is administered to reduce adiposity; the modulator is an osteoregulin antagonist and is administered to increase atherosclerotic plaque stability by increasing plaque calcification (e.g., to prevent stroke); or the modulator is an osteoregulin agonist wherein the agonist is administered to increase vascular flexibility by decreasing atherosclerotic plaque calcification.
An additional aspect of the invention features a method for producing a polypeptide of claim 3 or 4. The method comprises (a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell comprises a heterologous polynucleotide that encodes said polypeptide and said polynucleotide is operably linked to a promoter sequence; and (b) recovering said polypeptide. Preferably, the polynucleotide comprises SEQ ID NO: 1, 3, 5, or 7, or, in the case where the cell is a bacterial cell, a preferred polynucleotide comprises SEQ ID NO: 33 or SEQ ID NO: 45, or rat or mouse nucleic acid sequence encoding an osteoregulin polypeptide lacking the N-terminal signal sequence.
Those skilled in the art will fully understand the terms used herein in the description and the appendant claims to describe the present invention. Nonetheless, unless otherwise provided herein, the following terms are as described immediately below.
xe2x80x9cOsteoregulin activityxe2x80x9d is the negative regulation of bone mineralization, bone density, and/or bone formation. Thus, an osteoregulin functions to decrease bone area, bone mineral content, bone mineral density, and/or bone density. xe2x80x9cOsteoregulin activityxe2x80x9d also includes the negative regulation of the calcification of atherosclerotic plaques and the stimulation of increased adiposity. A polypeptide with xe2x80x9costeoregulin activityxe2x80x9d exhibits at least one of these activities.
An osteoregulin xe2x80x9cagonistxe2x80x9d refers to a molecule which intensifies or mimics the biological activity of an osteoregulin. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which increases the activity of an osteoregulin either by increasing the amount of osteoregulin present in a cell or by increasing the signaling of an osteoregulin polypeptide in its signal transduction pathway.
An xe2x80x9callelic variantxe2x80x9d is an alternative form of the gene encoding an osteoregulin. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to naturally-occurring deletions, additions, or substitutions of nucleotide. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
xe2x80x9cAlteredxe2x80x9d nucleic acid sequences encoding an osteoregulin include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide with at least one functional characteristic of an osteoregulin. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding an osteoregulin. The encoded protein may also be xe2x80x9caltered,xe2x80x9d and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent osteoregulin. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of the osteoregulin is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
xe2x80x9cAmplificationxe2x80x9d relates to the production of additional copies of a nucleic acid sequence. It is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
An osteoregulin xe2x80x9cantagonistxe2x80x9d refers to a molecule which inhibits or attenuates the biological activity of an osteoregulin. Antagonists may include proteins such as anti-osteoregulin antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which decreases the activity of an osteoregulin either by reducing the amount of osteoregulin present in a cell, or by decreasing the signaling of an osteoregulin polypeptide in its signal transduction pathway.
The term xe2x80x9cantibodyxe2x80x9d refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(abxe2x80x2)2, and Fv fragments, which are capable of binding an epitopic determinant. Antibodies that bind osteoregulin polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. The coupled peptide is then used to immunize the animal.
The term xe2x80x9cbiologically activexe2x80x9d refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, xe2x80x9cimmunologically activexe2x80x9d refers to the capability of the natural, recombinant, or synthetic osteoregulin, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
A xe2x80x9ccompositionxe2x80x9d comprising a given polynucleotide or amino acid sequence refers broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution.
xe2x80x9cConservative amino acid substitutionsxe2x80x9d are those substitutions that, when made, least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. Table A below shows amino acids which may be substituted for an original amino acid and which are regarded as conservative amino acid substitutions.
Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
The term xe2x80x9cderivativexe2x80x9d refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A xe2x80x9cfragmentxe2x80x9d is a unique portion of an osteoregulin or the polynudeotide encoding an osteoregulin which is identical in sequence to but shorter in length than the parent sequence. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from, or lack, certain regions of a molecule. For example, an osteoregulin polypeptide fragment may be the N-terminal signal sequence of an osteoregulin, such as amino acids 1-16 of SEQ ID NO: 2, 4, 6, or 8, or a fragment may contain an RGD motif.
The term xe2x80x9cidentityxe2x80x9d refers to a degree of complementarity. There may be partial similarity or complete identity. The word xe2x80x9csimilarityxe2x80x9d may substitute for the word xe2x80x9cidentity.xe2x80x9d A partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as xe2x80x9csubstantially similar.xe2x80x9d The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization, and the like) under conditions of reduced stringency. A substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency. This is not to say that conditions of reduced stringency are such that non-specific binding is permitted. Rather, reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% similarity or identity). In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.
The phrases xe2x80x9cpercent identityxe2x80x9d and xe2x80x9c% identity,xe2x80x9d as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison Wis.). CLUSTAL V is described in Higgins and Sharp CABIOS 5:151-153, 1989 and in Higgins et al. CABIOS 8:189-19, 1992. For pairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and xe2x80x9cdiagonals savedxe2x80x9d=4. The xe2x80x9cweightedxe2x80x9d residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the xe2x80x9cpercent similarityxe2x80x9d between aligned polynucleotide sequence pairs. Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-410, 1990), which is available from several sources, including the NCBI, Bethesda, MID, and at http://www.ncbi.nim.nih.lzov/13LAST/. The BLAST software suite includes various sequence analysis programs including xe2x80x9cblastn,xe2x80x9d that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called xe2x80x9cBLAST 2 Sequencesxe2x80x9d that is used for direct pairwise comparison of two nucleotide sequences. xe2x80x9cBLAST 2 Sequencesxe2x80x9d can be accessed and used interactively at http:/www.ncbi.nim. nih.lzov/gorf/b12.html. The xe2x80x9cBLAST 2 Sequencesxe2x80x9d tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the xe2x80x9cBLAST 2 Sequencesxe2x80x9d tool Version 2.0.9 (May 7, 1999) set at default parameters. Such default parameters may be, for example Matrix: BLOSUM62 Rewardfor match: 1 Penaltyfor mismatch: -2 Open Gap: 5 and Extension Gap.-2 penalties Gap x drop-off.-50 Expect 10 Word Size: 1 Filter: on. Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length disclosed by the sequences shown herein may be used to describe a length over which percentage identity may be measured. Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences encompassed by the invention that all encode substantially the same osteoregulin protein.
The phrases xe2x80x9cpercent identityxe2x80x9d and xe2x80x9c% identity,xe2x80x9d as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the hydrophobicity and acidity at the site of substitution, thus preserving the structure and function of the polypeptide. Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MegAlign(copyright) sequence alignment program (DNASTAR, Madison, Wis.). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty-3, window-5, and xe2x80x9cdiagonals savedxe2x80x9d=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the xe2x80x9cpercent similarityxe2x80x9d between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the xe2x80x9cBLAST 2 Sequencesxe2x80x9d tool Version 2.0.9 (May 7, 1999) with blastp set at default parameters. Such default parameters may be, for example, Matrix: BLOSUM62 Open Gap: 11 and Extension Gap: 1 penalties Gap x drop-off: 50 Expect: 10 Word Size: 3 Filter: on. Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number (e.g., 2, 4, 6, 8, or 10), or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
By a xe2x80x9chostxe2x80x9d is meant a transgenic cell (e.g., mammalian, bacterial, insect) or an animal (e.g., non-human mammal) that is transfected with, and capable of expressing, a heterologous polynudeotide.
xe2x80x9cHuman artificial chromosomesxe2x80x9d (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance.
The term xe2x80x9chumanized antibodyxe2x80x9d refers to an originally non-human antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
xe2x80x9cHybridizationxe2x80x9d refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of identity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the xe2x80x9cwashingxe2x80x9d step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68xc2x0 C. in the presence of about 6xc3x97SSC, about 1% (w/v) SDS, and about 100 pg/ml denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Generally, such wash temperatures are selected to be about 5xc2x0 C. to 20xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Press, Plainview, N.Y.; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of about 55-68xc2x0 C. in the presence of about 0.2-1.0xc3x97SSC and about 0.1% SDS, for 1 hour.
In general, hybridization reactions can be carried out at temperatures of about 65xc2x0 C., 60xc2x0 C., 55xc2x0 C., or 42xc2x0 C. may be used. SSC concentration may be varied from about 0.1 to 2xc3x97SSC, with SDS being present at about 0.1%. Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, denatured salmon sperm DNA at about 100-200 pg/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, is suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term xe2x80x9chybridization complexxe2x80x9d refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed between sequences present in solution or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides).
By xe2x80x9cisolated or purifiedxe2x80x9d is meant changed from the natural state xe2x80x9cby the hand of man.xe2x80x9d If a polynudeotide or polypeptide exists in nature, then it is xe2x80x9cisolated or purifiedxe2x80x9d if it is changed and/or removed from As original environment. For example, an xe2x80x9cisolated or purifiedxe2x80x9d polynucleotide is separated from other polynucleotide sequence with which it is associated in nature. For example, a cDNA sequence is removed from intronic sequence normally associated with the coding sequence contained in the cDNA. Such a sequence may be introduced into another cell for recombinant expression. However, polynucleotide sequences as found in cDNA libraries are excluded from what is meant by xe2x80x9cisolated or purified.xe2x80x9d An xe2x80x9cisolated or purifiedxe2x80x9d polypeptide is separated from at least one cellular component with which it is associated in nature. Preferably, the polypeptide is at least 60% free, more preferably, at least 75% free, and, most preferably, at least 90% from from other components.
xe2x80x9cOperably linkedxe2x80x9d refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter functions to regulate transcription or expression of the coding sequence. Generally, operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
xe2x80x9cPeptide nucleic acidxe2x80x9d (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
xe2x80x9cPolynucleotidexe2x80x9d generally refers to any RNA (e.g., mRNA), RNA-like, DNA (e.g., cDNA or genomic), or DNA like sequences, including, without limit, single-stranded, double-stranded, and triple-stranded sequence, senst or antisense strands, sequence generated using nucleotide analogs, hybrid molecules comprising RNA and DNA, and RNA or DNA containing modified bases. The polynucleotide can be naturally-occurring or synthesized.
The term xe2x80x9cpolypeptidexe2x80x9d refers to an amino acid sequence, oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. It includes amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modifications well known in the art (see, e.g., Proteinsxe2x80x94Structure and Molecular Properties, Ed. Creighton, W. H. Freeman and Co., New York, N.Y., 2nd Ed, 1993; Posttransiational covalent Modification of Proteins, Ed. Johnson, Academic Press, New York, N.Y., 1983; Seifter et al., Meth. Enzymol., 182: 626-46, 1990; and Rattan et al., Ann. NY Acad. Sci. 663: 48-62, 1992).
xe2x80x9cProbesxe2x80x9d refer to nucleic acid sequences encoding osteoregulins, their complements, or fragments thereof, which are used to detect identical, allellic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
xe2x80x9cPrimersxe2x80x9d are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length disclosed by the specification, including the tables, figures, and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in the literature (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Press, Plainview, N.Y. 1989; Ausubel et al., Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, New York, N.Y. 1987; and Innis et al., PCR Protocolsxe2x80x94A Guide to Methods and Application, Academic Press, San Diego, Calif. 1990). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose (e.g., Primer, Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).
Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software Is useful for the selection of PCR primer pairs and for the analysis of oligonucleotides and larger polynucleotides of up to nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas Tex.) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge Mass.) allows the user to input a xe2x80x9cmispriming library,xe2x80x9d in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user""s specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
A xe2x80x9csubstitutionxe2x80x9d refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
A xe2x80x9cvariantxe2x80x9d of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the xe2x80x9cBLAST 2 Sequencesxe2x80x9d tool Version 2.0.9, set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or greater sequence identity over a certain defined length. A variant may be described as, for example, an xe2x80x9callelicxe2x80x9d (as defined above), xe2x80x9csplice,xe2x80x9d xe2x80x9cspecies,xe2x80x9d or xe2x80x9cpolymorphicxe2x80x9d variant. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass xe2x80x9csingle nucleotide polymorphismsxe2x80x9d (SNPS) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
xe2x80x9cTransformationxe2x80x9d or xe2x80x9ctransfectionxe2x80x9d describes a process of genetic modification by which heterologous (i.e., foreign or exogenous) DNA enters and renders a recipient cell capable of expressing the heterologous DNA. Transformation may occur according to various methods well known in the art, for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment. The terms xe2x80x9ctransformed cellsxe2x80x9d or xe2x80x9ctransfected cellsxe2x80x9d include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed or transfected cells which express the inserted DNA or RNA for limited periods of time. All of such transformed or transfected cells are referred to as xe2x80x9ctransgenic.xe2x80x9d
A non-human mammal or an animal cell that is xe2x80x9cgenetically-modifiedxe2x80x9d is heterozygous or homozygous for a modification that is introduced into the non-human mammal or animal cell, or into a progenitor non-human mammal or animal cell, by genetic engineering. The standard methods of genetic engineering that are available for introducing the modification include homologous recombination, viral vector gene trapping, irradiation, chemical mutagenesis, and the transgenic expression of a nucleotide sequence encoding antisense RNA alone or in combination with catalytic ribozymes. Preferred methods for genetic modification to disrupt a gene are those which modify an endogenous gene by inserting a xe2x80x9cforeign nucleic acid sequencexe2x80x9d into the gene locus, e.g., by homologous recombination or viral vector gene trapping. A xe2x80x9cforeign nucleic acid sequencexe2x80x9d is an exogenous sequence that is non-naturally occurring in the gene. This insertion of foreign DNA can occur within any region of the osteoregulin gene, e.g., in an enhancer, promoter, regulator region, noncoding region, coding region, intron, or exon. The most preferred method of genetic engineering for gene disruption is homologous recombination, in which the foreign nucleic acid sequence is inserted in a targeted manner either alone or in combination with a deletion of a portion of the endogenous gene sequence.
By an osteoregulin gene that is xe2x80x9cfunctionally disruptedxe2x80x9d is meant an osteoregulin gene that is genetically modified such that the cellular activity of the osteoregulin polypeptide encoded by the disrupted gene is decreased or eliminated in cells that normally express a wild type version of the osteoregulin gene. When the genetic modification effectively eliminates all wild type copies of the osteoregulin gene in a cell (e.g., the genetically-modified, non-human mammal or animal cell is homozygous for the osteoregulin gene disruption or the only wild type copy of osteoregulin gene originally present is now disrupted), then the genetic modification results in a reduction in osteoregulin polypeptide activity as compared to an appropriate control cell that expresses the wild type osteoregulin gene. This reduction in osteoregulin polypeptide activity results from either reduced osteoregulin gene expression (i.e., osteoregulin mRNA levels are effectively reduced and produce reduced levels of osteoregulin polypeptide) and/or because the disrupted osteoregulin gene encodes a mutated polypeptide with reduced function or stability as compared to a wild type osteoregulin polypeptide. Preferably, the activity of osteoregulin polypeptide in the genetically-modified, non-human mammal or animal cell is reduced to 50% or less of wild type levels, more preferably, to 25% or less, and, even more preferably, to 10% or less of wild type levels. Most preferably, the osteoregulin gene disruption results in non-detectable osteoregulin activity.
By a xe2x80x9cgenetically-modified, non-human mammalxe2x80x9d containing a functionally disrupted osteoregulin gene is meant a non-human mammal that is originally produced, for example, by creating a blastocyst or embryo carrying the desired genetic modification and then implanting the blastocyst or embryo in a foster mother for in utero development. The genetically-modified blastocyst or embryo can be made, in the case of mice, by implanting a genetically-modified embryonic stem (ES) cell into a mouse blastocyst or by aggregating ES cells with tetraploid embryos. Alternatively, various species of genetically-modified embryos can be obtained by nuclear transfer. In the case of nuclear transfer, the donor cell is a somatic cell or a pluripotent stem cell, and it is engineered to contain the desired genetic modification that functionally disrupts the osteoregulin gene. The nucleus of this cell is then transferred into a fertilized or parthenogenetic oocyte that is enucleated, the embryo is reconstituted, and developed into a blastocyst. A genetically-modified blastocyst produced by either of the above methods is then implanted into a foster mother according to standard methods well known to those skilled in the art. A xe2x80x9cgenetically-modified, non-human mammalxe2x80x9d includes all progeny of the mammals created by the methods described above, provided that the progeny inherit at least one copy of the genetic modification that functionally disrupts the osteoregulin gene. It is preferred that all somatic cells and germline cells of the genetically-modified mammal contain the modification. Preferred non-human animals that are genetically-modified to contain a disrupted osteoregulin gene include rodents, such as mice and rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, and ferrets.
By a xe2x80x9cgenetically-modified animal cellxe2x80x9d containing a functionally disrupted osteoregulin gene is meant an animal cell, including a human cell, created by genetic engineering to contain a functionally disrupted osteoregulin gene, as well as daughter cells that inherit the disrupted osteoregulin gene. These cells may be genetically-modified in culture according to any standard method known in the art. As an alternative to genetically modifying the cells in culture, non-human mammalian cells may also be isolated from a genetically-modified, non-human mammal that contains an osteoregulin gene disruption. The animal cells of the invention may be obtained from primary cell or tissue preparations as well as culture-adapted, tumorigenic, or transformed cell lines. These cells and cell lines are derived, for example, from endothelial cells, epithelial cells, islets, neurons and other neural tissue-derived cells, mesothelial cells, osteocytes, lymphocytes, chondrocytes, hematopoietic cells, immune cells, cells of the major glands or organs (e.g., testicle, liver, lung, heart, stomach, pancreas, kidney, and skin), muscle cells (including cells from skeletal muscle, smooth muscle, and cardiac muscle), exocrine or endocrine cells, fibroblasts, and embryonic and other totipotent or pluripotent stem cells (e.g., ES cells, ES-like cells, and embryonic germline (EG) cells, and other stem cells, such as progenitor cells and tissue-derived stem cells). The preferred genetically-modified cells are ES cells, more preferably, mouse or rat ES cells, and, most preferably, human ES cells.
By an xe2x80x9cES cellxe2x80x9d or an xe2x80x9cES-like cellxe2x80x9d is meant a pluripotent stem cell derived from an embryo, from a primordial germ cell, or from a teratocarcinoma, that is capable of indefinite self renewal as well as differentiation into cell types that are representative of all three embryonic germ layers.
By xe2x80x9cmodulatesxe2x80x9d is meant increases or decreases (including a complete elimination).
Other features and advantages of the invention will be apparent from the following detailed description and from the claims. While the invention is described in connection with specific embodiments, it will be understood that other changes and modifications that may be practiced are also part of this invention and are also within the scope of the appendant claims. This application is intended to cover any equivalents, variations, uses, or adaptations of the invention that follow, in general, the principles of the invention, including departures from the present disclosure that come within known or customary practice within the art, and that are able to be ascertained without undue experimentation. Additional guidance with respect to making and using nucleic acids and polypeptides is found in standard textbooks of molecular biology, protein science, and immunology (see, e.g., Davis et al., Basic Methods in Molecular Biology, Elsevir Sciences Publishing, Inc., New York, N.Y.,1986; Hames et al., Nucleic Acid Hybridization, IL Press, 1985; Molecular Cloning, Sambrook et al., Current Protocols in Molecular Biology, Eds. Ausubel et al., John Wiley and Sons; Current Protocols in Human Genetics, Eds. Dracopoli et al., John Wiley and Sons; Current Protocols in Protein Science, Eds. John E. Coligan et al., John Wiley and Sons; and Current Protocols in Immunology, Eds. John E. Coligan et al., John Wiley and Sons). All publications mentioned herein are incorporated by reference in their entireties.